Negative selection, the process by which autoreactive T cells are removed from the maturation pathway, is a fundamental of self tolerance. We proposed that this process occurs through the mechanism of activation-induced apoptosis, a form of cellular "suicide", and we have found evidence to support this idea. In this research project, we will pursue the phenomenon of activation-induced cell death (AICD) from several perspectives, using in vivo and in vitro models of AICD. We will rigorously test the proposed relationship between AICD and negative selection in vivo, and investigate the development of susceptibility to AICD in maturing thymocytes. For these analyses we will take advantage of the DNA fragmentation that is characteristic of cells undergoing apoptosis. If negative selection is important in the development of self tolerance, and if AICD is the mechanism of negative selection, then defects in the AICD process should interfere with negative selection and contribute to some autoimmune disorders. We will examine this idea by using pharmacologic and physiologic inhibitors of AICD in vivo, and analyzing the effects of these agents on negative selection and the appearance of autoimmune disorders. Conversely, we will examine the effects of agents that are capable of inducing T cell-mediated autoimmune disfunction (eg, streptococcal cell wall) on the regulation of AICD and negative selection. To extend these findings, and to explore the cellular and molecular basis for AICD, we will take advantage of the phenomenon of AICD in T cell hybridomas, which we have shown to proceed via apoptosis. Using this in vitro system, we have identified the inhibitors to be employed in the in vivo experiments, and we will continue to screen for regulators of this process. Further, we have begun the dissection of the activation events required for entry into apoptosis following stimulation, and we will pursue this analysis in our proposed studies. We expect that some, but perhaps not all, of the cellular and molecular events that accompany activation will be required for AICD. We will therefore pursue our initial studies on the roles of the cell cycle, activation of protein kinase C, calcium influx, and the expression of the c-myc oncogene. The latter is controlled by the use of antisense oligodeoxynucleotides, and we will apply this analysis to other important T cell activation genes. We will complete and extend our findings in this area and relate our observations to the in vivo situation. Through a thorough understanding of the events surrounding AICD, we will learn how negative selection is sometimes bypassed to result in autoimmunity, and how such defects can be prevented